Welcome to a National Institutes of Health Clinical Center Department of Transfusion Medicine presentation on the cryopreservation of red blood cells. Blood group laboratories must often preserve rare red blood cells for later use. An important method for preserving these red blood cells is called droplet freezing. It is a procedure that uses liquid nitrogen to freeze red blood cells into tiny droplets. Droplet freezing is convenient because of how simple it is to thaw samples one only needs warm saline and a few minutes. The following materials are required to do droplet freezing.
Test tubes Fine tip transfer pipette 0.9 saline Autolet lancets Clamps Tweezers Funnel Metal canister Safety gloves and goggles 2.5 mL cryo vials with caps Liquid nitrogen holder Metal sieve Liquid nitrogen Polyvinyl Pyrrolidone freezing solution Cryostorage box And a centrifuge Droplet freezing can be divided into three main stages protecting, freezing, and thawing. Protecting This stage involves isolating the red blood cells and then treating them with a cryprotective agent. Begin with an anticoagulated whole blood sample. Isolate the red blood cells and wash them repeatedly in 0.9 saline until no hemolysis.
Is present in the supernatant. At this point, slowly add one volume of polyvinyl pyrrolidone freezing solution for every one volume of red blood cells. Gently mix. Let this mixture sit at room temperature for one hour. If polyvinyl pyrrolidone is not available, then it is possible to use an alternative freezing solution made from sucrose and dextrose. Nevertheless, polyvinyl pyrrolidone is more effective than sucrose and dextrose. Freezing Set out three metal canisters. Fill two canisters with liquid nitrogen. Place a metal sieve over the empty canister. Gently mix the sample and draw it into a finetipped transfer pipette.
The NIH Clinical Centers Guide to Blood Droplet Freezing
Hold the pipette vertically and 15 cm above the liquid nitrogen. Slowly release droplets into the liquid nitrogen. Do not release more than one droplet per second. If you release the droplets too quickly, then they will clump together. Also take caution to prevent air from entering the pipette. This will make droplets that are bloated with air bubbles. Use the metal sieve to collect the droplets. As long as the droplets remain sitting on the sieve over the liquid nitrogen, they should remain frozen. At this point, it is possible to remove suboptimal droplets with tweezers.
These are optimal droplets. Notice how they are uniform, smooth, and not clumped together. These are suboptimal droplets They are either too small, filled with air bubbles, or clumped together. Before transferring the droplets to a cryovial for longterm storage, puncture the cryovial’s cap 2 or 3 times. If small amounts of liquid nitrogen get into the vials, these holes will prevent the vials from exploding in storage. Chill the cryovial and funnel in liquid nitrogen. This will prevent the droplets from sticking and thawing on contact. Use the funnel to direct the droplets into the cryovial.
If droplets do stick to the funnel, moderate tapping may be necessary to dislodge the droplets. One of the liquid nitrogen canisters can be used as temporary storage until the all samples are frozen. When done, transfer the cryovials to longterm liquid nitrogen storage. At such low temperatures, these droplets may be stored for years. Thawing Drop the desired amount of droplets into 37C saline. Gently mix by pipetting up and down. Centrifuge the red blood cells and aspirate the supernatant to remove any hemolyzed cells. Continue washing with saline until the supernatants are clear.